Hidenori Sakuma

Clinicopathological significance of the CRTC3–MAML2 fusion transcript in mucoepidermoid carcinoma

Mucoepidermoid carcinoma is the most common primary malignancy of the salivary gland. We and others showed that CRTC1–MAML2 gene fusion was associated with favorable clinicopathological tumor features. Recently, a novel gene fusion, CRTC3–MAML2, was reported as a rare gene alteration in a case of mucoepidermoid carcinoma. However, its frequency and clinicopathological significance remains unclear. In all, 101 cases of mucoepidermoid carcinoma and 89 cases of non-mucoepidermoid carcinoma of the salivary gland were analyzed, and RNA was extracted from formalin-fixed, paraffin-embedded specimens. In the CRTC family, there have been three genes, CRTC1, CRTC2, and CRTC3. We developed reverse transcription-polymerase chain reaction (RT-PCR) assays for CRTC1–MAML2, CRTC2–MAML2, and CRTC3–MAML2 fusions. Clinicopathological data of the patients were obtained from their clinical records. Of 101 cases of mucoepidermoid carcinoma, 34 (34%) and 6 (6%) were positive for CRTC1–MAML2 and CRTC3–MAML2 fusion transcripts. However, in the 89 cases of non-mucoepidermoid carcinoma, neither transcript was noted. In the former cases, CRTC1–MAML2 and CRTC3–MAML2 fusions were mutually exclusive. The other fusion, CRTC2–MAML2, was not detected. We confirmed that the clinicopathological features of CRTC1–MAML2-positive mucoepidermoid carcinomas indicated an indolent course. CRTC3–MAML2-positive mucoepidermoid carcinomas also had clinicopathologically favorable features; all cases showed a less advanced clinical stage, negative nodal metastasis, no high-grade tumor histology, and no recurrence or tumor-related death after surgical resection of the tumor. It is interesting to note that patients with CRTC3–MAML2-positive tumors (mean 36 years of age) were significantly younger that those with the CRTC1–MAML2 fusion (55 years) and those with fusion-negative tumors (58 years). In conclusion, CRTC3–MAML2 fusion, which is mutually exclusive with CRTC1–MAML2 fusion and specific to mucoepidermoid carcinoma, may be detected more frequently than previously expected. Mucoepidermoid carcinomas possessing CRTC3–MAML2 fusion may be associated with favorable clinicopathological features and patients may be younger than those with CRTC1–MAML2 fusion or those with no detectable gene fusion.

Prognostic factors for keratocystic odontogenic tumor (odontogenic keratocyst): analysis of clinico-pathologic and immunohistochemical findings in cysts treated by enucleation

BACKGROUND The purpose of this study was to determine prognostic factors for the recurrence of keratocystic odontogenic tumors (KCOTs) following simple enucleation by examining clinico-pathologic and immunohistochemical findings. METHODS Following enucleation, the frequency of recurrence among 32 subjects diagnosed with KCOT was analyzed for tumor site, radiographic and histologic features, and immunopositivity for Ki-67 and p53. RESULTS Keratocystic odontogenic tumors in four out of 32 subjects (12.5%) recurred during the follow-up period (median: 33 months, range: 7-114 months). Three out of four subjects (75.0%) among recurrent group showed high expression of Ki-67 (LI >10%) in basal layer and four (4/28; 14.3%) among non-recurrence group (P = 0.025). Expression of p53 among non-recurrent group was observed in 11 subjects (11/28; 39.3%), and in three subjects (3/4; 75.0%) among the recurrent group (P = 0.295). Hazard risk for the recurrence of KCOT was 4.02 (95% CI 1.42-18.14) for high Ki-67 expression in the basal layer by the Cox proportional hazard model (P = 0.009). In our study, none of the other clinico-pathologic variables were associated with the recurrence of KCOT. CONCLUSION The results suggested that the evaluation of Ki-67 expression in KCOT at the time of pathological diagnosis might be helpful for consideration of appropriate adjunctive surgical procedures to avoid a recurrence and may serve as a prognostic marker.

Immunoglobulin VH gene analysis in gastric MALT lymphomas

The majority of gastric mucosa-associated lymphoid tissue (MALT) lymphomas are successfully treated with Helicobacter pylori eradication alone. However, certain subsets of these tumors are resistant to the eradication treatment. As API2-MALT1 fusion is a feature of one of these subsets, we divided gastric MALT lymphomas into three groups: eradication-responsive and API2-MALT1 fusion-negative (Group A), eradication-resistant and fusion-negative (Group B), and eradication-resistant and fusion-positive (Group C). To characterize further gastric MALT lymphomas, we analyzed VH genes, which do not change in the course of tumor progression, by extensive subcloning of the monoclonal PCR products of 45 cases. VH3-23 and VH3-30 were preferentially used in Group A tumors (14/23 cases, 61%) as compared with Group B (1/10 cases, 10%, P=0.0094) and Group C (2/12 cases, 17%, P=0.017). Tumors of Groups B and C used variegated VH fragments, and no dominant VH fragments were noted. Somatic mutation was detected in most of the cases. Ongoing mutation was detected in 3/45 cases (7%), when assessed according to strict criteria for a confirmed mutation. These findings suggest that inflammation-dependent tumors (Group A) may be derived from a highly restricted, probably H. pylori-associated, B cell subset and may not often progress to those that are inflammation-independent (Groups B and C). Although considered to be common in this tumor, ongoing mutation may be infrequent when assessed by strict criteria.

Analysis of API2-MALT1 fusion, trisomies, and immunoglobulin VH genes in pulmonary mucosa-associated lymphoid tissue lymphoma.

Pulmonary mucosa-associated lymphoid tissue lymphoma is unique in that chronic inflammation is rare and that API2-MALT1 fusion, resulting from t(11;18)(q21;q21), occurs frequently. In this study, we examined 20 cases for API2-MALT1 fusion using the multiplex reverse-transcription polymerase chain reaction and looked for trisomy 3, trisomy 18, and abnormalities of MALT1 and IGH genes using fluorescence in situ hybridization. In addition, we analyzed VH genes by subcloning of the monoclonal polymerase chain reaction products. Of 20 cases studied, we detected gene abnormalities in 16: API2-MALT1 fusion in 9, trisomy 3 in 5, trisomy 18 in 4, MALT1 abnormality in 13, and IGH abnormality in 1. MALT1 gene abnormalities were concordant with API2-MALT1 fusion or trisomy 18. One case showed API2-MALT1 fusion and trisomy 3. On detection of API2-MALT1 fusion and trisomies, we were able to divide our cases into 3 groups, API2-MALT1 positive, trisomy positive, and no detectable gene abnormality, suggesting that tumor development had processed along different genetic pathways. All 20 cases were analyzed for VH genes. Most of the VH genes selected by the lymphomas belonged to the VH3 family, but there was no restriction to any particular VH fragment. Of interest, VH genes were unmutated in 7 cases, suggesting that T-cell-independent extrafollicular B-cell maturation may be important in the development of this lymphoma. In addition, both mutated and unmutated tumor cases were found to carry the API2-MALT1 fusion and trisomy 3. This observation suggests that these gene abnormalities may occur in microenvironments found before or outside of follicular germinal centers.

Spontaneous regression of intraoral mucosa-associated lymphoid tissue lymphoma: Molecular study of a case

Mucosa‐associated lymphoid tissue (MALT) lymphoma presentation in the oral cavity is very rare. Reported herein is a case of intraoral MALT lymphoma of the minor salivary gland in a 70‐year‐old woman with Sjogren’s syndrome. Unexpectedly, a spontaneous clinically and histologically confirmed regression occurred 1 month after the tumor biopsy for diagnosis. Considering that salivary MALT lymphoma is associated with Sjogren’s syndrome and that the chronic inflammation caused by Sjogren’s syndrome persisted, it is hypothesized that the tumor clone might be present in the regressed lesion. Minimal residual tumor clone identical with the primary lesion was detected using the polymerase chain reaction (PCR) clonality assay for immunoglobulin heavy chain gene (IgH) rearrangement. No recurrence was clinically evident 38 months after the diagnosis. Spontaneous regression of MALT lymphoma should be examined at the molecular level in addition to clinical and histological evaluations. When minimal residual disease is detected, close follow up is necessary for early detection of the tumor relapse.

Risk of surgical glove perforation in oral and maxillofacial surgery.

Oral and maxillofacial surgery, which involves several sharp instruments and fixation materials, is consistently at a high risk for cross-contamination due to perforated gloves, but it is unclear how often such perforations occur. This study aimed to address this issue. The frequency of the perforation of surgical gloves (n=1436) in 150 oral and maxillofacial surgeries including orthognathic surgery (n=45) was assessed by the hydroinsufflation technique. Orthognathic surgery had the highest perforation rate in at least 1 glove in 1 operation (91.1%), followed by cleft lip and palate surgery (55.0%), excision of oral soft tumour (54.5%) and dental implantation (50.0%). The perforation rate in scrub nurses was 63.4%, followed by 44.4% in surgeons and first assistants, and 16.3% in second assistants. The odds ratio for the perforation rate in orthognathic surgery versus other surgeries was 16.0 (95% confidence interval: 5.3-48.0). The protection rate offered by double gloving in orthognathic surgery was 95.2%. These results suggest that, regardless of the surgical duration and blood loss in all fields of surgery, orthognathic surgery must be categorized in the highest risk group for glove perforation, following gynaecological and open lung surgery, due to the involvement of sharp objects.

Primary mucosa-associated lymphoid tissue lymphoma of the prostate: tumor relapse 7 years after local therapy.

Primary mucosa‐associated lymphoid tissue (MALT) lymphoma of the prostate is rare, and only five cases have been reported. Reported herein is a new case that has involved a 9 year follow up. A 79‐year‐old man was treated with transurethral resection (TUR) for a mass of the right prostatic lobe, and followed up under a diagnosis of benign prostatic hyperplasia with atypical lymphoid infiltration. Seven years later TUR was again performed for a right lobe mass. The lesion was diagnosed as a relapsed MALT lymphoma after detailed histological and immunoglobulin heavy chain gene analyses of the initial and relapsed lesions. Interestingly, lymphoepithelial lesions were observed only infrequently in this tumor. The API2‐MALT1 fusion, a gene alteration specific to MALT lymphoma, was absent. The patient had stage IA disease at the time of tumor relapse, and has been alive and well for the 2 years after the second TUR. The present case suggests that despite tumor recurrence, prostatic MALT lymphoma is indolent, and function‐preserving therapy is warranted.

Analysis of VH Gene Rearrangement and Somatic Hypermutation in Sjogren’s Syndrome and IgG4-Related Sclerosing Sialadenitis

IgG4‐related sclerosing sialadenitis is currently considered as an autoimmune disease distinct from Sjogren’s syndrome (SS) and responds extremely well to steroid therapy. To further elucidate the characteristics of IgG4‐related sclerosing sialadenitis, we analysed VH fragments of IgH genes and their somatic hypermutation in SS (n = 3) and IgG4‐related sclerosing sialadenitis (n = 3), using sialolithiasis (n = 3) as a non‐autoimmune control. DNA was extracted from the affected inflammatory lesions. After PCR amplification of rearranged IgH genes, at least 50 clones per case (more than 500 clones in total) were sequenced for VH fragments. Monoclonal IgH rearrangement was not detected in any cases examined. When compared with sialolithiasis, there was no VH family or VH fragment specific to SS or IgG4‐related sclerosing sialadenitis. However, rates of unmutated VH fragments in SS (30%) and IgG4‐related sclerosing sialadenitis (39%) were higher than that in sialolithiasis (14%) with statistical significance (P = 0.0005 and P < 0.0001, respectively). This finding suggests that some autoantibodies encoded by germline or less mutated VH genes may fail to be eliminated and could play a role in the development of SS and IgG4‐related sclerosing sialadenitis.

p53 and ki67 as biomarkers in determining response to chemoprevention for oral leukoplakia

BACKGROUND We performed a randomized controlled chemoprevention trial of oral leukoplakia by administrating a low dose of beta-carotene and vitamin C supplements. 17% of subjects in the experimental arm (4/23) demonstrated clinical remission (complete or partial response) at completion of the trial. The objective of this study was to determine whether baseline expression of p53 and ki67 demonstrated any differences between those responding or not responding to our intervention. A secondary objective was to elucidate any relationship between dietary factors and clinical responses. METHODS For this biomarker study, we included all subjects in the experimental group (n = 23) who were non-smokers. Among 16 who completed the trial for 1 year of supplementation, there were four responders and 12 non-responders at 1-year follow-up. Following immuno-staining for p53 and ki67, the percentage of positive cell nuclei were analyzed as labeling index (LI). RESULTS Expression of p53 was greater in basal layers than in para-basal layers. Mean para-basal LI of p53 was higher in non-responding (26.0) than in responding subjects (11.2) (P = 0.028). ki67 LIs were not significantly different in the two groups. CONCLUSIONS Expression of p53 was inversely related to clinical response to the supplements. Other biomarkers that may recognize subjects responsiveness to chemoprevention require further study.

Elucidating risk factors for oral leukoplakia affecting gingivae in Japanese subjects

Background: Clinicopathological studies have revealed some distinct anatomical variations in the clinical presentation of oral leukoplakia (OL). Screening studies have shown that gingival leukoplakia (GL) to be more common among the Japanese. Objective: A hospital-based case-control study was undertaken among patients diagnosed with OL to compare clinico-pathological data between GL and OL affecting other sites (n-GL). Methods: One hundred fifty-one subjects attending four city hospitals in Japan diagnosed with OL were recruited to this study. OL was diagnosed by World Health Organization criteria. They were interviewed for risk factors, clinical presentations were charted and investigated by patch testing for allergy to dental materials and by biopsy for dysplasia. Results: Eighty-eight with GL and 63 with n-GL were included in the study. GL lesions were predominantly white (93%) though n-GL leukoplakia had red (speckled) foci in 26%. Eighty percent of GL were seen in association with prosthesis or restorations. There were no significant differences by age or in tobacco and alcohol use in the two groups. Metal allergy test was positive in 58% of the GL but was not significantly higher compared to the n-GL (41%). Histopathological features showed that inflammatory cell infiltrate was predominant in n-GL group (p = 0.021). Higher dysplasia grades were also found in n-GL (p = 0.018). During follow-up, nine oral cancers (14%) were detected in n-GL group and among GL cases four developed oral carcinomas (5%) (p = 0.043). Cox hazard regression analysis revealed that after adjusting for age and sex, GL group was found to have a lower malignant risk, with a hazard ratio of 0.89 (95% CI: 0.51–1.04) when compared to n-GL. Conclusions: GL was more benign compared to n-GL in Japanese subjects, though risk factors appear similar. Further study is warranted to ascertain whether trauma may play an important role for hyperkeratinization of gingival tissues and possible evolution of GL to proliferative verrucous leukoplakia in later years.